DESCRIPTION: HIV-1 effectively evades the host immune responses and develops resistance to antiviral drugs. This creates the need for discovery of new therapeutic targets for intervention HIV-1 fusion with the host cell membrane is a key step leading to infection and is therefore an attractive target for developing antivirals. A number of peptide and small molecule inhibitors of HIV-1 fusion have been identified and a few are used in the clinic. However, the virus' ability to quickly acquire resistance to existing drugs justifies the development of novel antiretroviral strategies. Our recent finding that, contrary to common perception, HIV-1 enters target cells via an endocytic pathway suggests new avenues for fighting infection. This finding highlights the importance of host factors involved in endocytosis and vesicular trafficking for HIV-1 fusion. These cellular factors are attractive targets for small-molecule inhibitors, since these are unlikey to select for resistant HIV-1 variants. We propose to screen large chemical libraries for small molecule inhibitors of HIV-1 fusion. We have adapted and optimized for high-throughput screening (HTS) an enzymatic assay that directly measures virus-cell fusion and have designed counterscreens to eliminate false-positives and identify novel compounds that block HIV-1 entry/fusion. Top positive hits from the primary screen will be validated and selected compounds will be investigated and classified using a panel of novel functional and biochemical assays. Hits that block the fusion of HIV-1 and unrelated viruses entering cells through endocytosis are likely to target common entry pathways and can thus represent a new class of broad-spectrum inhibitors. We also hope to identify compounds exhibiting broad-spectrum virucidal activity against HIV-1 and other enveloped viruses. The proposed project is thus expected to identify novel HIV-1 fusion inhibitors, develop novel probes for studies of virus trafficking and suggest new cellular targets for therapy.